The cell division cycle is the fundamental biological process by which a cell grows, replicates its DNA and then divides to give two daughter cells. It includes four sequential phases, G1, S, G2, and M (see Pardee et al.; “Animal cell cycle”, Ann. Rev. Biochem., Vol. 47(1978), 715-750). DNA replication occurs in S phase, and replicated DNA is distributed to two genetically identical daughter cells in M phase. Between these two phases are gap1 (G1) and gap2 (G2). G1 is between M phase and S phase when a cell is responsive to extracellular stimuli and starting to grow. Gap2 is between S phase and M phase when the cell prepares for entry into mitosis. Lengths of the individual phases of the cell cycle can vary with cell type and with conditions. In addition, there is a fifth state known as G0 (or quiescence), into which cells may reversibly exit from the cell cycle but remain metabolically active.
The cell division cycle is regulated by the cyclin dependent kinases (CDKs), which belong to the family of serine/threonine kinases. For full activity, these kinases must form a complex with a member of the cyclin family of regulatory subunits and be phosphorylated on a specific threonine or serine residue (e.g., Thr 161 for cdk1). These active CDK/cyclin complexes then regulate cell cycle progression by phosphorylating their protein substrates whose activities are required at specific phases of the cell cycle. For example, in late S phase cyclin B is expressed and forms active CDK1/cyclin B complex, which is required for the entry of cells into M phase and considered as M phase promoting factor (see Kishimoto et al., “In vivo regulation of the entry into M-phase: initial activation and nuclear translocation of cyclin B/Cdc2”, Prog. Cell Cycle Res., Vol. 3 (1997), 241-249). This CDK1/cyclin B complex could phosphorylate several proteins including Histone H1, DNA polymerase alpha, RNA polymerase II, retinoblastoma tumor suppressor protein (Rb), lamin A, cAb1, nucleolin, which are involved in the early events of mitosis. In early G1, cyclin Ds are expressed and active CDK4/cyclin D and CDK6/cyclin D are formed, which then phosphorylate retinoblastoma tumor suppressor protein Rb (see Kato et al., “Induction of S phase by G1 regulatory factors”, Front. Biosci., Vol. 4 (1999), 787-792). In late G1, the cyclin Es are expressed and active CDK2/cyclin E complexes are generated, which also phosphorylate Rb. It is this sequential phosphorylation of Rb by CDK4/cyclin D, CDK6/cyclin D and CDK2/cyclin E that blocks its growth inhibitory effects. When Rb is hypophosphorylated, it tightly binds and blocks the activity of E2F, a trancription factor complex that is necessary for the expression of genes governing the G1 to S phase transition. When Rb is phosphorylated, its interaction with E2F is disrupted and E2F dependent transcription occurs. This allows the cell to pass the restriction point after which the cell no longer needs mitogenic stimulation to complete one cell cycle. In S phase, cyclin A(s) are expressed and active CDK2/cyclin A complexes are formed, which can phosphorylate the transcription factors B-Myb and E2F, and cdc6, a factor required for the initiation of DNA replication (see Kitagawa et al., “Phosphorylation of E2F-1 by cyclin A-cdk2”, Oncogene, Vol. 10 (1995), 229-236). The sequential activation of CDK/cyclin complexes and their ability to phosphorylate and thus regulate the action of other proteins allows a cell to progress from one completed phase of the cell cycle to the next, in the correct order.
Disruption in the function of CDKs and the order of cell cycle is implicated in the hyperproliferative diseases, such as cancer, psoriasis and rheumatoid arthritis (Kamb et al., “A Cell Cycle Regulator Potentially Involved in Genesis of Many Tumor Types,” Science, Vol. 264 (1994), 436-440).
For example, cyclin D1 is overexpressed in breast, esophageal, and squamous cell carcinomas (DelSal et al., “Cell Cycle and Cancer: Critical Events and the G1 Restriction Point,” Critical Rev. Oncogenesis, Vol. 71 (1996), 127-142) and overexpression of cyclin E is linked to a wide variety of solid tumors. The expression of CDK inhibitor p27, which is a substrate and also inhibitor of CDK2/cyclin E, is normally inhibited in prostate, colon and breast cancers, and the cellular levels of p27 are inversely correlated with the stage of disease (see Loda et al., “Increased Proteasome-dependent Degradation of the Cyclin Dependent Kinase Inhibitor p27 in Aggressive Colorectal Carcinomas,” Nature Medicine, Vol. 3 (1997), 231-234). In addition, genes encoding the CDK4 specific inhibitors of the p16 family frequently have deletions and mutations in sarcomas, leukemias, gliomas, familial melanoma and pancreatic, non-small cell lung, and head and neck carcinomas (see Nobori et al., “Deletions of the Cyclin Dependent Kinases 4 Inhibitor Gene in Multiple Human Cancers,” Nature, Vol. 368 (1994), 753-756). These data clearly confirm the unregulation of CDKs activity in oncogenesis.
The emerging data provide strong evidence for using compounds inhibiting CDKs, CDK2, and CDK4 in particular, as antiproliferative therapeutic agents. For example, BMS 387032 (Bristol-Myers. Squibb) is currently in Phase I clinical trials as an oncology chemotherapeutic (see Kim et al., “Discovery of Aminothiazole Inhibitors of Cyclin Dependent Kinase 2: Synthesis, X-ray Crystallographic Analysis, and Biological Activities”, J. Med. Chem., Vol. 45 (2002), 3905-3927) and is a selective ATP competitive inhibitor for CDK2 (IC50 of 48 nM). This compound induces cell cycle arrest and apoptosis, with concomitant inhibition of phosphorylation of CDK2 substrates such as retinoblastoma protein, demonstrating cytotoxicity in 40 human cell lines and showing antitumor activity in five tumor models, including P388 mouse leukemia, Cyclin E overexpressing transgenic mouse breast carcinoma, A2780 human ovarian carcinoma, Colo205 human colorectal carcinoma, and A431 human squamous cell carcinoma. Likewise, flavopiridol has shown potent activity against CDKs, arresting the cell cycle in the G1 and G2 phases by direct inhibition of CDK2, CDK4, and CDK1 and by down regulation of cyclins D1, D3, and B, inhibiting various human cancer cell lines and showing activity in prostate melanoma, breast cancer and NSCLC tumor xenograft models. Flavopiridol was administered by IV infusion to patients with advanced tumors in a Phase I study (see Thomas et al., “Phase I Clinical and Pharmacokinetic Trial of the Cyclin Dependent Kinase Inhibitor Flavopiridol”, Cancer Chemotherapy & Pharmacology, Vol. 50 (2002), 465-72). A partial response was observed in a patient with renal cancer and 3 minor responses were seen in one of each patients with colon or prostate cancer and non-hodgkin's lymphoma. A phase II trial investigated the use of flavopiridol and paclitaxel with or without co-administration of cisplatin in the treatment of 54 patients with advanced solid tumors. Among the 51 evaluable patients, a total of 1 complete, 1 partial and 1 minor response and 21 cases of stable disease were reported (see Koroukis et al., “Flavopiridol in Untreated or Relapsed Mantle-cell Lymphoma: Results of a Phase II Study of the National Cancer Institute of Canada Clinical Trials Group”, Journal of Clinical Oncology, Vol. 21 (2003), 1740-45; and Schwartz et al., “Phase II Study of Cyclin Dependent Kinases Inhibitor Flavopiridol Administered to Patients with Advanced Gastric Carcinoma”, Journal of Clinical Oncology, Vol. 19 (2001), 1985-93). Indisulam (E-7070) is a novel sulfonamide agent in Phase II with Eisai for head and neck cancer, solid tumor, CRC and NSLC and has demonstrated a unique antitumor spectrum in vitro and excellent antitumor activity in vivo affecting multiple cell-cycle checkpoints. E-7070 suppresses the expression of cyclin E and phosphorylation of CDK2 by blocking cell cycle progression at several points, including G1/S and G2/M transition. In vivo studies showed its activity against various human cancers, such as colon carcinomas HCT116, LS174T, SW620 and HCT15 and NSCLC PC-9 (see Ova et al. “Discovery of Novel Antitumor Sulphonamides Targeting G1 Phase of the Cell Cycle”, J. Med. Chem., Vol. 42 (1999), 3789-3799; and Ozawa et al., “E7070, A Novel Sulphonamide Agent With Potent Antitumor Activity in vitro and in vivo”, Eur. J. of Cancer, Vol. 37 (2001), 275-282). Roscovitine (Cyc 202) (Cyclacel) is in clinical development for various cancers, inhibiting CDK2 and to a lesser extent also CDK1. This compound is cytotoxic in HCT116 human colon carcinoma cells and has been shown in patients to induce apoptosis by acting on G1/S or early checkpoint (see McClue et al., “In Vivo and In Vitro Antitumor Properties of The Cyclin Dependent Kinase Inhibitor CYC 202 (R-roscovitine)”, International Journal of Cancer, Vol. 102(5) (2002), 463-468). UCN-01 (Kyowa Hakko Kogyo) is a non-specific kinase inhibitor with activity against PKCs and CDKs. Preclinical data indicates that the main potential of UCN-01 is in the treatment of leukemias, in particular CLL, but the compound has been advanced clinically in renal cell carcinoma and pancreatic cancer. Furthermore, this compound has shown antitumor activity in patients with melanoma and lymphoma (see Senderowicz et al., “Phase I Trial of Infusion UCN-01, a Novel Protein Kinase Inhibitor, in Patients With Refractory Neoplasms”, Annals of Oncology, Vol. 9 (Suppl. 2) (1998), 111).
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However, the demand is still increasing for small molecule compounds that can be readily made and are potent inhibitors of one or more CDKs or CDK/cyclin complexes. Because CDK1 may serve as an M phase promoting factor and govern the G2 phase of the cell cycle, and CDK2/cyclin E controls the early G1 phase of the cell cycle, there is a need for effective and specific inhibitors of CDK1 and/or CDK2 for treating one of more types of tumors.
Also, inhibition of CDKs may prevent progression in the cell cycle in normal cells and limit the toxicity of cytotoxics which act in S phase, G2 or Mitosis. Such disruption of the cell cycle or normal proliferating cells should therefore protect proliferating cells such as hair follicles and epithelial mucosa from the effects of cytotoxic agents and thereby provide a potent treatment for side effects associated with cancer chemo- and radiotherapies.